The present invention relates generally to fluorescent dye compounds that are useful as molecular probes. In particular, the present invention relates to fluorescent cyanine dye compounds that are mobility modified for use in nucleic acid sequencing reactions.
The advent of automated four-color Sanger-type DNA sequencing has revolutionized the speed with which stretches of DNA can be reliably sequenced. In four-color Sanger-type DNA sequencing, a single-stranded target DNA of interest is hybridized with a complementary primer and the primer enzymatically extended with a DNA polymerase in the presence of a mixture of 2xe2x80x2-deoxyribonucleotides capable of supporting continuous primer extension (e.g., dATP, dGTP, dCTP and dTTP or dUTP) and a mixture of four labeled terminators. Each of the terminators is labeled with a different, spectrally distinguishable fluorescent label and terminates primer extension at a single type of template nucleotide. A mixture of terminators is used such that a termination event is achieved at each type of template nucleotide. The product of this primer extension or sequencing reaction is a nested set of labeled primer extension products in which the 3xe2x80x2-terminal nucleotide is identifiable by the color of its fluorescent label. These products are then electrophoretically separated, typically in a single gel lane or capillary, and the sequence of the target DNA determined from the colors of the resultant electrophoresis bands.
To avoid ambiguities in determining the sequence of the target DNA, the dyes used to label the primer extension products should either impart no electrophoretic mobility shifts on the products or impart uniform mobility shifts. However, in most instances, different types of dyes impart vastly different electrophoretic mobility shifts. Since the dyes must be spectrally distinguishable from one another, dyes having different structures, and hence quite different imparted electrophoretic mobility shifts, must be used. While sets of terminators that impart primer extension products with similar mobility shifts are available, rationally designing such sets of xe2x80x9cmobility matchedxe2x80x9d terminators is currently virtually impossible. Rather, the sets are obtained through empirical trial and error. To date, no methods exists whereby one can predictably alter the electrophoretic mobilities imparted by terminators labeled with desirable dyes without altering the spectral properties of the dyes and/or jeopardizing the abilities of the labeled terminators to act as substrates for polymerizing enzymes. Accordingly, these are objects of the present invention.
These and other objects are furnished by the present invention, which in one aspect provides cyanine dye compounds having a mobility-modifying moiety that permits the electrophoretic mobilities of polynucleotides labeled with the dyes to be adjusted or tuned in a predictable fashion.
Cyanine dyes are a well-recognized class of fluorescent molecules which generally comprise first and second parent heteroaromatic ring systems covalently linked together via a methine, polymethine or cyclic alkylene bridge. The dyes may be homodimers, in which the first and second parent heteroaromatic ring systems are both members of the same class, or they may be heterodimers, in which the first and second parent heteroaromatic ring systems are both members of different classes. The parent ring systems may be optionally substituted with one or more substituents which can serve to alter the spectral, chemical and/or physical properties of the dyes.
The present invention concerns the class of cyanine dyes in which both parent heteroaromatic rings belong to the class of rings generally referred to as benzazoles/benzazoliums. The mobility-modifying cyanine dyes of the invention generally comprise: (i) a first parent benzazole/benzazolium heteroaromatic ring system that is substituted at the heteroaromatic ring nitrogen with a linking moiety; (ii) a second parent benzazole/benzazolium heteroaromatic ring system that is substituted at the heteroaromatic ring nitrogen with a mobility-modifying moiety; and (iii) a bridge linking the first and second parent benzazole/benzazolium rings via their respective C-2 carbons. The first and second parent benzazole/benzazolium ring systems may be the same or different, and may be optionally substituted with one or more of the same or different substituent groups. Preferably, both parent benzazole/benzazolium ring systems are the same or different substituted or unsubstituted indoline/indolinium ring systems. Depending upon the particular application, the linking moiety can be used to conjugate, preferably by way of covalent attachment, the mobility-modifying dyes of the invention to other molecules or substances.
Quite significantly, since the mobility-modified and linking moieties are located at opposing ends of the cyanine dye (i.e., on different heteroaromatic rings), nucleosides/tides and/or nucleoside/tide analogs labeled with the mobility-modifying cyanine dyes of the invention, e.g., labeled 2xe2x80x2-deoxyribonucleoside-5xe2x80x2-triphosphates and labeled terminating ribonucleoside-5xe2x80x2-triphosphates (e.g., 2xe2x80x2,3xe2x80x2-dideoxyribonucleoside-5xe2x80x2-triphosphates), retain high activity as substrates for DNA polymerizing enzymes, making the mobility-modifying dyes ideal for use in fluorescence-based nucleic acid sequencing applications. Moreover, since the electrophoretic mobilities of polynucleotides labeled with the mobility-modifying dyes can be predictably tuned to match those labeled with other dyes, the mobility-modifying dyes of the invention are ideal for use in 4-color fluorescence-based nucleic acid sequencing reactions, as sets of dyes having matched mobilities in addition to desirable spectral and biological properties can be readily obtained.
Virtually any known cyanine dye can be mobility-modified according to the principles of the invention. Thus, parent heteroaromatic ring systems of which the dyes of the invention can be comprised include, but are not limited to, the substituted and unsubstituted benzazole/benzazolium rings comprising the cyanine, merocyanine and styryl dyes described in U.S. Pat. Nos. 5,486,616, 5,569,587, 5,569,766 and 5,627,027; the substituted and unsubstituted benzazole/benzazolium rings comprising the asymmetric cyanine dyes described in U.S. Pat. Nos. 5,321,130, 5,410,030, 5,436,134, 5,534,416, 5,582,977, 5,658,751, 5,656,449, and 5,863,753; and the substituted and unsubstituted benzazole/benzazolium rings comprising the various sulfonated cyanine dyes described in Tu et al., 1998, Nucl. Acids Res. 26(11):2797-2802, the disclosures of which are incorporated herein by reference. Additional substituted and unsubstituted benzazole/benzazolium ring systems of which the mobility-modifying cyanine dyes may be comprised are described in Brooker et al., 1945, xe2x80x9cAbsorption Spectra of Dyes with Heteroaromatic Nucleixe2x80x94Color and Constitution. Part VII. Intepretation of Absorptions of Dyes Containing Heterocyclic Nuclei of Different Basicities,xe2x80x9d J. Am. Chem. Soc. 67:1875-1889 (in particular at page 1878), the disclosure of which is incorporated herein by reference.
The mobility-modifying moiety comprises a pendant group bearing a plurality of charges through substitution with one or more of the same or different charged substituents. The pendant group can be any moiety capable of being substituted with the desired number of charged substituents, but is typically a group having the structure xe2x80x94D-Dxe2x80x2, where D is (C1-C6) alkyldiyl or 2-6 membered heteroalkyldiyl; and Dxe2x80x2 is (C1-C6) alkyl, 2-6 membered heteroalkyl, (C5-C14) aryl, (C5-C14) arylaryl, 5-14 membered heteroaryl or 5-14 membered heteroaryl-heteroaryl. When D is heteroalkyldiyl, it must be attached to the benzazole/benzazolium ring nitrogen atom via an alkyldiyl group. Preferred amongst the various D groups is (C1-C6) alkyleno, particularly (C1-C6) alkanos such as methano (xe2x80x94CH2xe2x80x94), ethano (xe2x80x94CH2xe2x80x94CH2xe2x80x94), propano (xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94), etc.
The polarity of the charged substituents substituting the pendant group depends upon the direction of the desired electrophoretic mobility shift. When an increase in electrophoretic mobility is desired, anionic substitutents should be used. When a decrease in electrophoretic mobility is desired, cationic substiutents should be used. When multiple charged substitutents are used they can be the same or different, and can even be of mixed polarities, although in most instances all of the charged substituents will have the same polarity.
The number of charged substituents substituting the pendant group depends upon the desired net charge of the mobility-modifying moiety, which in turn depends upon the identity of the charged substituent and the degree of mobility modification necessary. The charged substituents may be any substituent group having a net charge at the desired pH of use (typically pH 6 to 10). Suitable cationic substituents include, by way of example and not limitation, permanent cations such as quaternary ammoniums, especially those of the formula xe2x80x94N+RRR, where each R is independently (C1-C6) alkyl, and cations derived from bases. Permanent cations or cationic substituents that are derived from strong bases (xe2x80x9cstrong cationic substituentsxe2x80x9d), such as those having a pKa of about 8 or greater, are preferred, as these strong cationic substituents are completely ionized at the pHs commonly employed in biological assays such as nucleic acid sequencing reactions.
Suitable anionic substituents include groups having a pKa of 6 or less, and include by way of example and not limitation, xe2x80x94C(O)Oxe2x88x92, xe2x80x94P(O)(Oxe2x88x92)2, xe2x80x94P(O)(OH)(Oxe2x88x92), xe2x80x94Oxe2x80x94P(O)2(Oxe2x88x92), xe2x80x94S(O)2Oxe2x88x92 and xe2x80x94Oxe2x80x94S(O)2Oxe2x88x92 (including any associated counterions). Anionic substituents that are derived from strong acids (xe2x80x9cstrong anionic substituentsxe2x80x9d), such as those having a pKa of 3 or less, are preferred, as these strong anionic substituents are completely ionized at the pHs commonly employed in biological assays such as nucleic acid sequencing reactions. Preferred amongst the strong anionic substituents are xe2x80x94S(O)2Oxe2x88x92 and xe2x80x94Oxe2x80x94S(O)2Oxe2x88x92.
In addition to the desired charged substituents, the pendant group may be further substituted with one or more additional uncharged substituents. Such uncharged substituents can serve a variety of purposes, e.g., to increase the water solubility of the mobility-modifying dye, to decrease non-specific binding of the mobility-modifying dyes and/or to decrease the interactions between chromophores of multiply labeled compounds, thereby decreasing quenching of fluorescence.
The bridge joining the two parent heteroaromatic ring systems can be any type of bridge commonly used to join the parent heteroaromatic ring systems of cyanine dyes. Preferably, the bridge permits electron delocalization. Electron-delocalizing bridges useful for linking the heteroaromatic rings of the dyes include, but are not limited to, methine, polymethine, squarine and cyclic alkene bridges. The bridges may be optionally substituted with one or more of the same or different substituents that typically serve to increase the chemical and/or photostability of the dye and/or increase its quantum yield.
The linking moiety has the structure xe2x80x94Lxe2x80x94LG, where L is a linker and LG is a linking group that can be used to conjugate, preferably by way of covalent attachment, the mobility-modifying cyanine dye to another compound or substance, such as a protein, nucleosideltide, polynucleotide, polymer, particle etc. The identity of linking group LG will depend upon the nature of the desired conjugation. For example, the conjugation may be: (i) mediated by ionic interactions, in which case linking group LG is a charged group; (ii) mediated by hydrophobic interactions, in which case linking group LG is a hydrophobic moiety; (iii) mediated by covalent attachment, in which case linking group LG is a reactive functional group (Rx) that is either capable of forming a covalent linkage with another complementary functional group (Fx) or is capable of being activated so as to form a covalent linkage with complementary functional group Fx; or (iv) mediated through the use of pairs of specific binding molecules, such as biotin and avidin/streptavidin, in which case linking group LG is one member of the pair, e.g., biotin.
The linking group LG is attached to the benzazole/benzazolium ring nitrogen via linker L. Depending upon the application, linker L can be hydrophobic, hydrophilic, long or short and/or rigid, semirigid or flexible. Regardless of the identity of the linker L, in order to avoid adversely affecting the spectral properties of the cyanine dye chromophore, it must be attached to the benzazole/benzazolium nitrogen atom via an alkyldiyl group.
In another aspect, the invention provides labeled conjugates comprising a mobility-modifying cyanine dye according to the invention and another molecule or substance. The mobility-modifying cyanine dye is conjugated to the other molecule or substance, typically via covalent attachment, through linking group LG as previously described. Once conjugated, the dye provides a convenient fluorescent label for subsequent detection. The dyes of the invention can be used to fluorescently label a wide variety of molecules and substances, including amino acids, proteins, antibodies, enzymes, receptors, nucleosides/tides, nucleic acids, carbohydrates, lipids, steroids, hormones, vitamins, drugs, metabolites, toxins, organic polymers, etc. The dyes can also be used to label particles such as nanoparticles, microspheres or liposomes. The molecule or substance may be labeled with one or more mobility-modifying cyanine dyes of the invention, which may be the same or different.
In one preferred embodiment, the labeled conjugate is a labeled nucleoside/tide or nucleoside/tide analog. The dye may be conjugated to either the sugar or nucleobase moiety of the receptive nucleosideltide or nucleoside/tide analog, but is usually conjugated to the nucleobase moiety.
The labeled nucleoside/tide or nucleosideltide analog may be enzymatically incorporable, in which case it may be conveniently used in conjunction with a template nucleic acid, a primer and appropriate polymerizing enzymes to enzymatically generate labeled polynucleotides. A particularly preferred class of enzymatically-incorporable labeled nucleoside/tides and nucleoside/tide analogs are labeled terminators, as such terminators can be conveniently used in Sanger-type sequencing reactions to generate labeled polynucleotide sequencing fragments having defined gel electrophoretic mobilities.
Alternatively, the labeled nucleoside/tide or nucleoside/tide analog may be synthetically incorporable, such as a labeled nucleosidic or non-nucleosidic phosphoramidite synthesis reagent. Such reagents can be conveniently used in conjunction with standard solid phase oligonucleotide synthesis reagents and supports to label synthetic polynucleotides and/or polynucleotide analogs at their 3xe2x80x2-terminus, their 5xe2x80x2-terminus and/or at one or more internal positions with mobility-modifying dyes of the invention.
In another aspect, the invention provides methods of using the dyes of the invention to sequence a target nucleic acid. The method generally comprises forming a series of differently-sized primer extension products that are labeled with a dye of the invention, separating the series of differently-sized labeled extension products, typically based on size, and detecting the separated labeled extension products based on the fluorescence of the label. The sequence of the target nucleic acid is then assembled according to known techniques.
The series of differently-sized labeled extension products can be conveniently generated by enzymatically extending a primer-target hybrid according to well-known methods. For example, the series of labeled extension products can be obtained using a primer labeled with a dye or dye pair of the invention and enzymatically extending the labeled primer-target hybrid in the presence of a polymerase, a mixture of enzymatically-extendable nucleotides or nucleotide analogs capable of supporting continuous primer extension and at least one, typically unlabeled, terminator that terminates primer extension upon incorporation (e.g., a 2xe2x80x2,3xe2x80x2-dideoxyribonucleoside-5xe2x80x2-triphosphate). Alternatively, the series of labeled extension products can be obtained using an unlabeled primer and enzymatically extending the unlabeled primer-target hybrid in the presence of a polymerase, a mixture of enzymatically-extendable nucleotides or nucleotide analogs capable of supporting continuous primer extension and at least one terminator labeled with a dye of the invention. In either embodiment, the polymerase serves to extend the primer with enzymatically-extendable nucleotides or nucleotide analogs until a terminator is incorporated, which terminates the extension reaction. Once terminated, the series of labeled extension products are separated, typically based on size, and the separated labeled extension products detected based on the fluorescence of the labels. The sequence of the target is then obtained via conventional means.
In a particularly advantageous embodiment of this method, a mixture of four different terminators are used in a single extension reaction. Each different terminator is capable of terminating primer extension at a different template nucleotide, e.g., a mixture of 7-deaza-ddATP, ddCTP, 7-deaza-ddGTP and ddTTP or ddUTP, and is labeled with a different, spectrally-resolvable fluorophore, where at least one of the fluorophores is a mobility-modifying dye according to the invention. According to this embodiment, an unlabeled primer-target nucleic acid hybrid is enzymatically extended in the presence of, a polymerase, a mixture of enzymatically-extendable nucleotides or nucleotide analogs capable of supporting continuous primer extension and a mixture of the four different, labeled terminators. Following separation based on size, a series of separated labeled extension products is obtained in which the emission properties (i.e., color) of each separated extension product reveals the identity of its 3xe2x80x2-terminal nucleotide. In a particularly preferred embodiment, all of the labeled terminators are excitable using a single light source.
Alternatively, terminators may be used in the absence of enzymatically-extendable nucleotides. In this instance, the primer is extended by only a single base. Again, the primer may be labeled or, alternatively, one or more of the terminators may be labeled. Preferably, a mixture of four different labeled terminators is used, as described above. These xe2x80x9cmini sequencingxe2x80x9d embodiments are particularly useful for identifying polymorphisms in chromosomal DNA or cDNA.
In yet another aspect, the invention provides mobility-matched sets of labeled terminators and/or polynucleotide primers that can be conveniently used in Sanger-type sequencing reactions to generate sequencing ladders having matched electrophoretic mobilities. For 4-color nucleic acid sequencing applications, one or several conventional cyanine dyes having the desired spectral properties can be selected and the respective comparative mobilities of polynucleotide fragments labeled therewith obtained. The cyanine dyes can then be simply mobility-modified according to the principles taught herein to tune the electrophoretic mobilities of polynucleotides labeled therewith as necessary to obtain sets of dyes that are mobility-matched. A preferred set of mobility-matched terminators includes Compounds 31, 32, 33 and 34 (see Section 5.10, infra). A preferred set of mobility-matched polynucleotide primers includes primers labeled with the dye chromophores of Compounds 31, 32, 33 and 34.
In a final aspect, the invention provides kits comprising the mobility-modifying cyanine dyes and/or labeled conjugates of the invention and reagents useful for labeling molecules and/or for performing assays such as nucleic acid sequencing.
The mobility-modifying cyanine dyes of the invention provide significant advantages over currently available cyanine dyes. Because the mobility-modifying moiety does not significantly alter the spectral properties of the cyanine dye chromophore, these dyes are useful in virtually any applications that utilize fluorescent dyes. However, owing to their ability to predictably alter the electrophoretic mobilities of polynucleotides labeled therewith, the mobility-modifying dyes of the invention provide the ability to create mobility-matched sets of fluorescent dyes for applications involving the electrophoretic separation of labeled polynucleotides, such as automated nucleic acid sequencing. In particular, mixed dye sets (i.e., dyes with different structures) may be conveniently employed in automated sequencing applications due to the ability to match the respective mobilities of polynucleotides labeled therewith according to this invention. Moreover, enzymatically-incorporable nucleoside/tides, enzymatically-incorporable nucleoside/tide analogs and terminators labeled with the mobility-modifying dyes of the invention retain high enzymatic activity with the polymerases commonly employed in automated nucleic acid sequencing methods, including thermostable polymerases such as AMPLITAQ(copyright) DNA polymerase FS (PE Biosystems, Foster City, Calif.).